1. Field of the Invention
This invention relates to a chemical agent useful in the treatment of pulmonary emphysema. More particularly, this invention relates to a covalent complex (or conjugate) of a water soluble polymer which may be a polysaccharide or a polyol with human alpha-l-proteinase inhibitor, to a process for producing the covalent complex (or conjugate) of a polysaccharide or a polyol with alpha-l-proteinase inhibitor, optionally in the presence of catalase enzyme, to a pharmaceutical preparation comprising the covalent complex (or conjugate) of a polysaccharide or a polyol with alpha-l-proteinase inhibitor, and to a method for treating pulmonary emphysema comprising administering to a human patient a therapeutically effective amount of the complex (or conjugate) or pharmaceutical preparation according to the invention.
Alpha-l-proteinase inhibitor (abbreviated ".alpha..sub.1 PI") is a glycoprotein having a molecular weight of 53,000 determined by sedimentation equilibrium centrifugation. The glycoprotein consists of a single polypeptide chain to which several oligosaccharide units are covalently bonded. Human alpha-l-proteinase inhibitor has a role in controlling tissue destruction by endogenous serine proteinases. A genetic deficiency of alpha-l-proteinase inhibitor, which accounts for 90% of the trypsin inhibitory capacity in blood plasma, has been shown to be associated with the premature development of pulmonary emphysema. The degradation of elastin associated with emphysema probably results from a local imbalance of elastolytic enzymes and the naturally occurring tissue and plasma proteinase inhibitors. Alpha-l-proteinase inhibitor inhibits human pancreatic and leukocyte elastases. See Pannell et al, Biochemistry, 13, 5339 (1974); Johnson et al, Biochem. Biophys. Res. Commun., 72, 33 (1976); Del Mar et al, Biochem, Biophys. Res. Commun., 88, 346 (1979); and Heimburger et al, Proc. Int. Res. Conf. Proteinase Inhibitors, 1st, 1-21 (1970).
2. Description of the Prior Art
Coan et al, U.S. Pat. No. 4,379,087, disclose a method for separating alpha-l-proteinase inhibitor from blood plasma or blood plasma fractions which contain the proteinase inhibitor. An aqueous solution of the blood plasma fraction is held at a pH of about 6.5-8.5 and at a temperature of about 2.degree.-50.degree. C., and for a period of about 0.2-24 hours and then mixed with a polycondensed polyglycol (e.g. polyethylene glycol) in the proportion of about 10-15 grams of polyglycol per 100 ml of aqueous solution containing the blood plasma fraction. The mixture may be held at temperature of about 2.degree.-10.degree. C. for a period of about 1-24 hours. Next, the pH of the mixture is adjusted to about 4.6-5.7 to selectively precipitate unwanted proteins from the solution without precipitation of alpha-l-proteinase inhibitor. Finally, alpha-l-proteinase inhibitor is separated from solution and purified further.
Other processes for the production of alpha-l-proteinase inhibitor have been reported. Pannell et al, Biochemistry, 13, 5439 (1974), mentioned above, disclose a process wherein albumin-poor blood plasma was pooled and fractionated with solid ammonium sulfate. The resulting precipitate was purified in a four-step procedure involving albumin removal using a Sepharose-Blue Dextran adsorption column, ammonium sulfate fractionation of the most active fractions from the first step, and two DEAE-cellulose chromatography separations.
Saklatvala et al, Biochem. J., 157, 339 (1976) disclose a process to obtain alpha-l-proteinase inhibitor by fractionating human plasma using ammonium sulfate and chromatographing the resulting precipitate on DEAE-cellulose. The 0.5M NaCl extract therefrom was applied to a concanavalin A-Sepharose column and eluted with alpha-D-methyl glucopyranoside. The eluate was again applied to a DEAE-cellulose column and an eluate containing alpha-l-proteinase inhibitor was obtained using 0.0-0.2M NaCl.
Musiani et al, Biochemistry, 15, 798 (1976) disclose the use of 50% aqueous ammonium sulfate to separate an alpha-l-proteinase inhibitor from blood plasma which was solubilized and subjected to successive chromatographic separations using DEAE in exchanger, concanavalin A-Sepharose, Sephadex G-100 and an immuno adsorbent columns to yield purified alpha-l-proteinase inhibitor.
Kress et al, Preparative Biochemistry, 3 (6), 541 (1973), disclose the large scale purification of alpha-l-proteinase inhibitor from human plasma using 80% ammonium sulfate aqueous solution, the precipitate from which treatment was solubilized, dialyzed and chromatographed on DEAE-cellulose. The resulting concentrate was again dialyzed and gel-filtered on Sephadex G-100 and the alpha-l-proteinase inhibitor containing fractions were chromatographed twice on DE-52 cellulose.
Glaser et al, Preparative Biochemistry, 5 (4), 333 (1975), isolated alpha-l-proteinase inhibitor from Cohn Fraction IV-1 in 30% overall yield by chromatographing the Cohn Fraction IV-1 on DEAE-cellulose, QAE-Sephadex, concanavalin A-Sepharose and Sephadex G-150.
Hao et al, Proceedings of the International Workshop on Technology for Protein Separation and Improvement of Blood Plasma Fractionation, 1977, Reston, Virginia, disclosed an integrated plasma fractionation system based on the use of polyethylene glycol (PEG) to obtain proteins distributed in four PEG fractions using 0-4% PEG, 4-10% PEG, 10-20% PEG and 20% PEG. Alpha-l-proteinase inhibitor was among the several proteins isolated in the 20% PEG fraction.
Stabilization and modification of enzymes and other proteins by covalent attachment to carbohydrates and polyethylene glycol has been reported. Marshall and Rabinowitz, Arch. Biochem. Biophys., 167, 77 (1975) and J. Biol. Chem., 251, 1081 (1976), noting earlier reports that glycoproteins (mostly enzymes) often show unusual stability characteristics compared with carbohydrate-free proteins, the former being less sensitive to heat and other denaturing conditions and more resistant to proteolysis, disclose the preparation of soluble enzyme-carbohydrate conjugates by coupling (by means of covalent attachment) trypsin, .alpha.-amylase and .beta.-amylase to cyanogen bromide activated dextran. The resulting covalent conjugates displayed marked resistance to heat inactivation and denaturation, increased half-life, and reduction in loss of activity under conditions favoring antolysis.
Vegarud et al, Biotechnol. Bioeng., 17, 1391 (1975) and Christensen et al, Process Biochemistry, 25 (July/August 1976), report the results of experiments carried out with "natural" glycoproteins as well as the "artificial" protein-glycoconjugates (produced by the cyanogen bromide method which have shown that glycosated enzymes are more stable towards heat inactivation by heat and proteases than the corresponding non-glycosated preparations.
Chaplin et al, Biotech. Bioeng., XXIV, 2627 (1982), disclose soluble conjugates of pepsin and carboxypeptidase A prepared by covalent linkage of the enzyme to an amino derivative of dextran having specific activities close to those of the native enzymes and having increased temperature and pH stabilities.
Tam et al, Proc. Natl. Acad. Sci., 73 (6), 2128 (1976), disclose a complex between soluble dextran and human hemoglobin, produced by two alternative methods involving cyanogen bromide (alkylation) and dialdehyde coupling chemistry, which is cleared through the kidneys and removed from circulation much more slowly than free hemoglobin in rabbits.
Hoylaerts et al, Thromb. Haemostas, (Stuttgart), 49 (2), 109 (1983), and Ceustermans et al, J. Biol. Chem., 257 (7), 3401 (1982), disclose covalent complexes of high affinity heparin fragments of low molecular weight and high affinity heparin with antithrombin-III having increased half-life compared with the uncomplexed heparin fragments and heparin and resulting in a 30-fold longer life time of Factor Xa inhibitory activity in plasma as compared with that of free intact heparin.
Bjork et al, FEBS Letters, 143 (1), 96 (1982), disclose covalent complexes formed by covalent attachment of antithrombin to high affinity heparin oligosaccharides, obtained by vitrous acid treatment of heparin, wherein the heparin oligosaccharide components have reactive aldehyde functions which form a Schiff's base with the amino group of any neighboring lysine residue of the protein.
Abuchowski et al, J. Biol. Chem., 252 (11), 3578 and 3582 (1977), disclose the modification of proteins, specifically, bovine serum albumin and bovine liver catalase, by the covalent attachment thereto of nonimmunogenic methoxyproylene glycols of 1900 daltons (PEG-1900, Union Carbide Corp.) and 500 daltons (PEG-5000, Union Carbide Corp.) using cyanuric chloride (2,4,6-trichloro-s-triazine) as the coupling agent. The modified bovine serum albumin exhibited a blood circulating life in rabbits similar to native bovine serum albumin except that it was not removed from circulation by the eventual development of antibodies.
Also, the modified bovine serum albumin exhibited substantial changes in properties, such as solubility, electrophoretic mobility in acrylamide gel, ion exchange chromatography, and sedimentation, as compared with the unmodified protein. Rabbits were immunized by the intravenous administration of PEG-1900-catalase. The intravenous antiserum/antibodies did not yield detectable antibodies against PEG-1900-catalase or native catalase whereas the intramuscular antiserum contained antibodies to PEG-1900-catalase and native catalase. PEG-5000-catalase did not react with either antiserum. PEG-1900-catalase and PEG-5000-catalase retained 93% and 95%, respectively, of their enzymatic activity and PEG-5000-catalase resisted digestion by trypsin, chymotrypsin and a protease from Streptoenyces griseus. PEG-1500-catalase and PEG-5000-catalase exhibited enhanced circulating lives in the blood of acatalasemic mice during repetitive intravenous injection and no evidence was seen of an immune response to injections of the modified enzymes.
Ashihara et al, Biochem. Biophys. Res. Commun., 83 (2), 385 (1978), disclose the modification of E. coli L-asparginase with activated polyethylene glycol (PEG-5000, PEG-1900, and PEG-750) to obtain products having varying levels of enzyme amino group substitution by means of covalent attachment of the polyethylene glycol to the enzyme amino groups. The modification of asparginase to 73 amino groups out of the total 92 amino groups in the molecule with PEG-5000 gave rise to a complete loss of the binding ability towards anti-asparginase serum from rabbits and retained the enzymatic activity (72) and hand versitivity against trypsin.
Koide et al, FEBS Letters, 143 (1), 73 (1982), disclose the preparation of polyethylene glycol-modified streptokinase by covalently attaching the glycol and the enzyme. The resulting modified streptokinase had a complete loss of antigenicity but had retention of its enzymatic activity.
O'Neill et al, Biotechnol. Bioeng., 13, 319 (1971) disclose the covalent attachment of the enzyme, chymotrypsin, to dextran and to DEAE-cellulose using 2-amino-4,6-dichloro-s-triazine as the coupling agent. Determination of the activity of the preparations showed that chymotrypsin attached to the soluble substrate had a considerably higher activity towards both casein and anti-tyrosine ethyl ester than did chymotrypsin attached to DEAE-cellulose. Both of the conjugates had increased relative stability compared with native chymotrypsin as determined by incubating at 40.degree. C. followed by assaying with acetyl-tyrosine ethyl ester (ATEE).